Lipoproteins in the living body are classified into high-density lipoprotein (hereinafter abbreviated as HDL), low-density lipoprotein (hereinafter abbreviated as LDL), very low-density lipoprotein (hereinafter abbreviated as VLDL), and chylomicron (hereinafter abbreviated as CM) according to their specific gravity. Each class of the lipoproteins has a considerably different function in vivo mainly depending on the type of the apoprotein and also has a different lipid composition. It is known that, of these lipoproteins, HDL is involved in the removal action of cholesterol accumulated in cells to receive cholesterol from tissues including arterial walls and is a risk prevention factor for various arterial scleroses, including coronary arteriosclerosis, and therefore, its level in blood is a useful index for predicting the onset of arteriosclerotic diseases.
The conventional methods for measuring cholesterol in HDL (hereinafter abbreviated as HDL cholesterol) consist of two steps: a fractionation operation by an ultracentrifugation method, an immunochemical method, an electrophoresis method, or a precipitation method, and cholesterol determination. However, the fractionation operations are complicated and time-consuming and also have a problem in terms of safety. Thus, these measurement methods involving the fractionation operations are extremely inefficient and are not suited for practical use.
Various measurement methods have been reported in recent years to solve the above problems. Known examples of the methods include: a method for the specific fractional determination of HDL cholesterol, which involves reacting serum or plasma with a cholesterol esterase and a cholesterol oxidase in a buffer solution comprising the above enzymes as well as a bile acid salt, a bile acid derivative, or dioctylsulfosuccinate (see patent document 1) and a method for measuring HDL cholesterol, which involves reacting serum with pancreas-derived cholesterol esterase and cholesterol oxidase in a buffer solution comprising the enzymes, a surfactant belonging to the group of bile acids, and a nonionic surfactant at a specific pH and a specific temperature (see patent document 2). In the method described in patent document 2, the reaction of LDL cholesterol with the enzymes proceeds first and HDL cholesterol can then be measured. However, these measurements require long time and have not always been measurement methods specific for HDL cholesterol.
Known examples of methods for measuring HDL cholesterol by aggregating lipoproteins other than HDL include: a method using a reagent for aggregating lipoproteins other than HDL, such as dextran sulfate, a divalent metal salt, and a chemically modified enzyme (see patent document 3); a measurement method using a reagent forming a complex with lipoproteins other than HDL, such as a polyanion, and a surfactant not dissolving lipoproteins, such as a polyoxyethylene-polyoxypropylene condensate, (see patent document 4); a measurement method using a polyanion, such as dextran sulfate, a divalent metal salt, a specific nonionic surfactant, and albumin different from the albumin derived from a sample (see patent document 5); and a method for measuring HDL cholesterol in serum or plasma, which involves treating serum or plasma with a solution comprising a lipoprotein fractionating agent (a combination of a polyanion, such as dextran sulfate, and a divalent cation, such as magnesium ion), reacting the resultant mixture with a cholesterol esterase and a cholesterol oxidase in the presence of an anionic surfactant (an alkylsulfonic acid, or a bile acid or a derivative thereof) without subjecting the mixture to solid-liquid separation, and measuring a formed hydrogen peroxide (see patent document 6).
Known examples of methods for aggregating HDL cholesterol without aggregating lipoproteins other than HDL include: a method for measuring HDL cholesterol in a biological sample, which involves reacting the biological sample with pancreas-derived cholesterol esterase and cholesterol oxidase in the presence of a bile acid or a salt thereof and albumin, and measuring a compound consumed or formed by the enzymatic reaction (see patent document 7); and a method for measuring HDL cholesterol in a specimen, which involves reacting the specimen with a lipoprotein lipase preferentially acting on an HDL fraction and/or a cholesterol esterase and a cholesterol oxidase in the presence of a nonionic surfactant with an HLB value of 16 or more, having reaction selectivity on the HDL fraction (see patent document 8). Also known is a method which involves preferentially converting cholesterol in lipoproteins other than HDL into hydrogen peroxide with acyl polyoxyethylene sorbitan ester; eliminating a formed hydrogen peroxide; and then enzymatically measuring HDL cholesterol by adding a polyoxyethylene alkyl ether (see patent document 9).
Further, known are: a method for measuring HDL cholesterol in a specimen, which involves reacting the specimen with i) a cholesterol ester hydrolase and a cholesterol oxidase, or ii) a cholesterol ester hydrolase, an oxidized coenzyme, and a cholesterol dehydrogenase in an aqueous medium comprising a nonionic surfactant, a polyanion, and albumin, and measuring a formed hydrogen peroxide or reduced coenzyme (see patent document 10); and a method for measuring HDL cholesterol, which involves reacting a specimen with i) a cholesterol ester hydrolase and a cholesterol oxidase, or ii) a cholesterol ester hydrolase, an oxidized coenzyme, and a cholesterol dehydrogenase in an aqueous medium comprising a quaternary ammonium salt or amine having a specific structure and a polyanion, and measuring a formed hydrogen peroxide or reduced coenzyme (see patent document 11).
In a case where a specific substance in a specimen is measured by an optical technique, there is a problem is that for a specimen derived from a patient with liver/biliary tract disease, abnormal lipoprotein (LpX) in which phospholipids and free cholesterol are increased appears, and the LpX has an optical influence on the measurement system to provide an incorrect measurements. To avoid the optical influence, a method is commonly known which involves increasing the concentration of salts in the reaction solution to eliminate turbidity due to water-insoluble proteins. However, in HDL cholesterol measurement, the presence of a high concentration of salts may lead to a reduced specificity to HDL cholesterol and further a deactivation of contained enzymes.